The ability to introduce foreign genes into mammalian cells has proven to be a powerful tool both in the research laboratory and for the commercial production of biologicals. Foreign genes are commonly introduced either on plasmid or viral vectors or by co transfection with such vectors. Several different methods have been used to introduce foreign genes including calcium phosphate precipitation, protoplast fusion, electroporation and viral infection. For many combinations of vectors, transfection methods, and mammalian cell lines, stable transfection is a rare event. Methods for the selection of cells containing foreign DNA are usually necessary. Therefore, selectable markers have been incorporated into many vectors.
There are a number of different selective methods for transfected DNA in mammalian cells. These methods may be divided into two classes based on the conferred phenotype. The first class consists of selection relating to dominant drug resistant markers. Markers in this class include resistance to neomycin (G418) (Southern and Berg, 1982), hygromycin B (Sugden et al., 1985) and mycophenolic acid (Mulligan and Berg, 1985). The second class consists of selection relating to complementation of an auxotrophic mutation. Several examples of this approach include the thymidine kinase (tk) gene (Wigler et al., 1977), the hypoxanthine guanine phosphoribosyl transferase (hgprt) gene (Mulligan and Berg, 1985) and the dihydrofolate reductase gene (dhfr) (Kaufman and Sharp, 1982).
The selective systems mentioned above have found broad applicability to the introduction of foreign DNA into a variety of mammalian cell types. However, there are limitations to each of the individual approaches. For example, some cell lines are natively resistant to G418: others grow poorly in mycophenolic acid even in the presence of the vector; and still others have a reduced growth rate under hygromycin B selection. Selection methods using neomycin and hygromycin B require high concentrations (greater than 100 ug/ml) and therefore are relatively expensive. Hygromycin B is also unstable in cell culture medium necessitating frequent refeeding in order to maintain selection.
Selection methods relying upon the complementation of auxotrophic mutations also have drawbacks. In contrast to dominant marker systems, further modification to the recipient cell is required. These systems also require the isolation of rare tk deficient, hgprt deficient or dhfr deficient cells This limits these approaches to cell lines where such mutants already exist or cell lines with high cloning efficiencies such that appropriate mutants may be isolated.
Aside from the drawbacks of known selective systems, it is sometimes necessary to independently introduce several different genes into a single cell line. Therefore, it is desirable to have several independent selective systems.
A new selective approach should have the following properties. Preferably, the selective marker is not normally present in the recipient cell line. Therefore, the recipient cell line does not have to be modified by, for example, mutation to remove a native property, and the usefulness of the marker does not depend upon the ability to create such a mutation. Most preferably the selective marker is not present in mammalian cells so that the selective system may be used in connection with mammalian cells. Further, the gene encoding the selective marker preferably is relatively small in order to facilitate introduction into vectors with limited "available space." The products used in the selective system should be stable in cell culture medium. Finally, the products used in the selective system should be relatively inexpensive. These and other objects are achieved according to the invention.